Reactive oxygen species (ROS) are a class of small molecules that play important roles in human health, aging, and disease. Imbalances in the production and consumption of ROS cause oxidative stress and damage of proteins, lipids, and nucleic acids, which can in turn lead to functional decline of tissues and organs over time and contribute to major diseases ranging from cancer to cardiovascular disorders to neurodegenerative diseases. At the same time, emerging data shows that one particular ROS, hydrogen peroxide (H2O2), can also be produced for physiological signaling purposes to mediate processes, from wound healing to neurotransmission, by regulating cell growth, differentiation, and migration pathways in a manner akin to the canonical small-molecule signal nitric oxide (NO). We are developing and applying new chemical tools for molecular imaging of H2O2 with the long-term goal of understanding how and in what context this oxygen metabolite contributes to normal physiology, aging, and disease. Specific aims for this competitive renewal submission include developing new probes that will allow chemoselective imaging of H2O2 with subcellular resolution, creating new imaging agents that can be used to monitor H2O2 fluxes in living organisms by near-IR fluorescence or bioluminescence modalities, and applying these and related chemical tools for elucidating the contributions of H2O2 production to signaling and stress pathways in neural stem cells and neurons.